Methods of using gap junctions as therapeutic targets for the treatment of degenerative disorders of the retina

ABSTRACT

The disclosure provides methods of treating a condition of the retina by administering an inhibitor of connexin 36 and/or an inhibitor of connexin 45 to a subject with a retinal condition. This disclosure further provides compositions for the treatment of a retinal condition which include an inhibitor of connexin 36 and/or an inhibitor of connexin 45.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application62/011,354, filed Jun. 12, 2014, which is incorporated herein in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number R01EY007360 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in the ASCII text file, named as31144sequence_SB25.txt of 12 KB bytes, created on Jun. 10, 2014, andsubmitted to the United States Patent and Trademark Office via EFS-Web,is incorporated herein by reference.

BACKGROUND OF THE DISCLOSURE

In addition to the intrinsic mechanisms underlying primary cell death,intercellular communication appears to play a major, but presentlyunclear, role in so-called secondary cell death (Andrade-Rozental A F,et al., Brain Res. Rev 32:308-315 (2000)). Damage in the central nervoussystem (CNS) leads to the death of a limited cohort of vulnerable cells,which, in turn, pass toxic molecules via gap junctions (GJs) to coupledneighbors. There is now substantial evidence that cells that areclustered and can thereby communicate via GJs tend to die en mass undera broad range of neurodegenerative conditions (Frantseva et al., J.Cereb. Blood Flow Metab. 22:453-462 (2002); Cusato et al., Cell DeathDiffer 13:1707-1714 (2006); Lei et al., Br J Ophthalmol. 93:1676-1679(2009); Wang et al., J Neurophysiol 104:3551-3556 (2010). In thisscheme, GJs act as portals for the passage of apoptotic signals frominjured cells to those to which they are coupled, which can ultimatelybe the cause of most cell loss (Kermer et al., Cell Tissue Res298:383-395 (1999); Perez Velazquez et al., Neuroscientist 9:5-9 (2003);Decrock et al., Cell Death Differ 16:524-536 (2009); Belousov andFontes, Trends Neurosci 36:227-236 (2013)).

There is increasing evidence that GJs are involved in variousneurodegenerative ocular disorders, including ischemic retinopathy andglaucoma (Krysko, Apoptosis 10:459-469 (2005); Malone, Glia 55:1085-1098(2007); Das et al., Biochem Biophys Res Commun 373:504-508 (2008); Kerret al., J Clin Neurosci 18:102-108(2011); Danesh-Meyer et al., Brain135:506-520 (2012)). The topography of neuronal loss in the inner retinaseen with these pathologies often includes both a diffuse, but clusteredpattern suggesting that dying retinal ganglion cells (RGCs) influenceneighboring cells, resulting in secondary neuronal degeneration(Levkovitch-Verbin, Invest Ophthalmol Vis Sci 42:975-982 (2001); Lei etal., Br J Ophthalmol. 93:1676-1679 (2009); Vander et al., Curr Eye Res37:740-748 (2012)). The GJ-mediated secondary cell death, or so-called“bystander effect”, has also been implicated in the programmed celldeath in the developing retina. Like in the adult, dying cells indeveloping retina are spatially clustered into distinct networks (Cusatoet al., Cell Death Differ 13:1707-1714 (2003); de Rivero Vaccari et al.,J. Neurophysiol 98:2878-2886 (2007)). Dopamine, which is a modulator ofGJ communication, as well as the GJ blockers octanol and carbenoxolonesignificantly reduce the rate of programmed and induced cell death inyoung retinas and the clustering of the remaining dying cells (Varellaet al., J Neurochem 73:485-492 (1999); Cusato, Cell Death Differ13:1707-1714 (2003)).

Amacrine cells (ACs) form the largest cohort of retinal neurons,comprising over 30 distinct morphological subtypes that subserve complexsynaptic interactions in the inner plexiform layer (IPL), which arelargely responsible for the diverse physiological properties expressedby RGCs (Demb J B, et al., Vis Neurosci. 29:51-60 (2012)). Studies ofglaucomatous human retinas have reported an apparent delayed orsecondary degeneration of amacrine cells subsequent to RGC cell loss(Schwartz Eur J Ophthalmol Suppl 3:S27-31 (2002); Kielczewski et al.,Invest Ophthalmol Vis Sci 46:3188-3196 (2005); Moon et al., Cell TissueRes. 320:51-59 (2005)). However, whether ACs are adversely affected inglaucoma remains unclear as conflicting experimental results have beenreported (Kielczewski J L, et al., Invest Ophthalmol Vis Sci. 46:3188-96(2005); GA, Barnett N L., et al. Clin Experiment Ophthalmol. 39:555-63(2011); Moon J I, et al., Cell Tissue Res. 320:51-9 (2005); Jakobs T C,et al., J Cell Biol. 171:313-25 (2005)).

One explanation for the discrepant findings may be the difficulty inclearly identifying ACs and thereby measuring their loss. For example,in addition to RGCs, displaced amacrine cells (dACs) comprise about 50%of the neurons found in the GCL of the mouse retina (Schlamp C L, etal., Mol Vis. 19:1387-96 (2013)) and no single labeling method canprovide complete coverage due to their wide morphological diversity.Interestingly, it has been reported that 16 of the 22 morphologicalsubtypes of RGCs in the mouse retina are coupled to ACs (Volgyi B, etal., J Comp Neurol. 512:664-87 (2009)). This extensive coupling suggeststhat GJ-mediated secondary cell death would likely progress from RGCs totheir coupled AC neighbors or vice versa. A downregulation of calretinin(CR), calbindin (CB), and choline acetyltransferase (ChAT) have beenreported in ischemic retinas (Dijk and Kamphius, Brain Res. 1026:194-204(2004); Bernstein and Guo, Invest Ophthalmol Vis Sci 52:904-910 (2011);Lee et al., Apoptosis 11:1215-1229 (2011)), suggesting that the loss ofthe AC immunoreactivity may be due to reduced protein detection ratherthan cell death.

Changes in the expression and function of certain connexin subtypes inCNS have been reported under a variety of pathological conditions(Rouach et al., Biol Cell. 94:457-475 (2002); Petrash-Parwez et al., J.Comp Neurol 479:181-197 (2004); Eugenin et al., J. Neuroimmune Pharmacol7:499-518 (2012); Kerr et al., J. Clin Neurosci 28:102-108 (2012)). Inaddition, the conductance of GJ hemichannels, related to their connexinmakeup, appears related to their ability to support bystander cell death(Kameritsch et al., Cell Death Dis 4:1-9 (2013)).

The conductance of GJs, based on their connexin makeup, appears relatedto their ability to support bystander cell death (Kameritsch P, et al.,Cell Death Dis. 4:e584 (2013)). In addition, changes in the expressionof certain connexin subtypes in CNS have been reported under a varietyof pathological conditions (Kerr N M, et al., Exp Neurol. 234:144-52,(2012); Rouach N, et al., Biol Cell. 94:457-75 (2002); Petrasch-ParwezE, et al., J Comp Neurol. 479:181-97 (2004); Eugenin E A, et al., JNeuroimmune Pharmacol. 7:499-518 (2012)).

Thus, the degree to which a particular GJ contributes to secondary celldeath is likely dependent on which of the different types of connexinsubunits it expresses as well as the insult condition. The fact that atleast three connexin subtypes are expressed in the IPL of the retinaraises the notion that different cohorts of GJs, based on their connexinprofile, may be responsible for secondary cell death in the inner retinaarising from different primary insults.

In contrast, some studies have reported that GJs may actually protectcells. Evidence for this “good Samaritan” role include the findings thatGJ inhibitors can induce apoptosis (Lee et al., Anat Cell Biol 44:25-34(2006); Hutnik et al., Invest Ophthalmol Vis Sci 49:800-806 (2008)) andthat deletion of GJ connexins can increase neuronal loss (Naus et al.,Cell Commun Adhes 8:325-328 (2001); Striedinger et al., Eur J Neurosci22:605-6016 (2005)). It has been posited that GJs are portals by whichhealthy cells provide dying neighbors with rescue signals or that thecoupled syncytium can dilute toxic substances (Krysko et al., PLoS One8:e57163 (2005)). Apoptotic conditions induce various changes in thestructure of GJs, including phosphorylation of connexins (Lin et al.,Exp Eye Res 85:113-122 (2007)), suggesting that the connexin makeup of aGJ may be a critical factor in determining its contribution to celldeath or survival.

The retina displays arguably the highest expression of GJs in the CNS,which are widely distributed amongst the five neuronal types and expressa variety of connexin subunits (Bloomfield and Volgyi, Nat Rev Neurosci10:495-506 (2009)). GJ-mediated secondary cell death has been implicatedin retinal neuron loss seen under a number of degenerative conditions,including retinitis pigmentosa, glaucoma, and ischemia (Ripps, Exp EyeRes 74:327-336 (2002); Das et al., Biochem Biophys Res Commun373:504-508 (2008)). On the other hand, deletion of connexins havefailed to increase the survivability of cone photoreceptors in a mousemodel of retinitis pigmentosa (Kranz et al., PLoS One 8:e57163 (2013))and has been reported to increase cell loss after retinal trauma(Striedinger et al., Eur J Neurosci 22:605-6016 (2005)), suggesting thatGJs can in fact be neuroprotective.

Thus, the role of retinal GJs in cell death and survival, and in thedevelopment or worsening of ocular conditions, remains unclear.

BRIEF SUMMARY OF THE DISCLOSURE

Disclosed herein are methods for treating a condition of the retina, byadministering an inhibitor of connexin 36 and/or an inhibitor ofconnexin 45 to a subject with a retinal condition. In some embodiments,both an inhibitor of connexin 36 and an inhibitor of connexin 45 areadministered. The condition of the retina can be selected, for example,from glaucoma, macular degeneration, retinitis pigmentosa, diabeticretinopathy and retinal ischemia.

In any of the above methods, the inhibitor can be selected from anantisense polynucleotide directed to connexin 36 messenger ribonucleicacid (mRNA), an antisense polynucleotide directed to connexin 45 mRNA,and combinations thereof. Preferred antisense polynucleotide inhibitorsare those that selectively bind the sequence of SEQ ID NO: 1 or SEQ IDNO: 2. The antisense polynucleotide can be complementary to all of or aportion of connexin 36 mRNA and/or connexin 45 mRNA. The antisensepolynucleotide can be the exact complement of all or a portion ofconnexin 36 mRNA and/or connexin 45 mRNA. The antisense polynucleotidecan hybridize to connexin 36 mRNA and/or connexin 45 mRNA with a meltingtemperature of greater than 20° C., 30° C. or 40° C. under physiologicalconditions.

Alternatively, the inhibitor can be a small molecule inhibitor.Exemplary small molecule inhibitors include 18-Beta-glycyrrhetinic acid(18Beta-GA or 18β-GA) and meclofenamic acid (MFA).

Any of the above methods can include repeat administration of theinhibitor or inhibitors for a period of one week to one year. Any of theabove methods can further include topical administration, such as a dropto be administered to the eye, or intraocular injection.

Further disclosed herein are pharmaceutical compositions for treatmentof a retinal condition. The compositions can include an inhibitor ofconnexin 36 and/or an inhibitor of connexin 45. In one example, theinhibitor or inhibitors can be selected from an antisense moleculedirected to connexin 36 mRNA, an antisense molecule directed to connexin45 mRNA, and combinations thereof. In another example, the compositionincludes a small molecule inhibitor of connexin 36 and/or a smallmolecule inhibitor of connexin 45. The small molecule inhibitor orinhibitors can be selected, for example, from 18-Beta-glycyrrhetinicacid (18Beta-GA) and meclofenamic acid (MFA). Any of the disclosedcompositions can be formulated for intraocular injection or topicaladministration to the eye.

Further disclosed herein are uses of any of the disclosed compositionsfor the treatment of a condition of the retina. In some examples, thecondition of the retina can be selected from glaucoma, maculardegeneration, retinitis pigmentosa, diabetic retinopathy and retinalischemia.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1I. Injection of cytochrome C (CytC) into single cells resultsin GJ-mediated apoptosis of neighboring coupled cells. A-F,Representative photomicrographs of uncoupled (A-C), and coupled (D-F)RGCs in whole mount retina injected with the mixture of Neurobiotin (NB)and CytC to visualize GJ coupling and to induce cell apoptosis,respectively. C, Overlay of panels (A), and (B) shows apoptosis of theimpaled RGC (asterisk), but not any neighboring cells. F, Overlay ofpanels (D) and (E) shows that apoptosis was spread amongst neighboringcells coupled to impaled RGC. G-I, Representative confocal image ofcoupled Müller cells in retinal vertical section, one of which(asterisk) was injected with NB+ CytC (G) and then labeled withanti-caspase 3 antibody (H) to detect apoptotic cells. (I), Overlay ofpanels (G) and (H) shows spread of death in coupled neighboring Müllercells. Scale bars=20 μm (A-C and G-I) and 50 μm (D-F).

FIGS. 2A-2E. N-methyl-D-aspartate (NMDA)-induced excitotoxic cell deathis significantly reduced by gap junction blockers. (A, C), NMDA-inducedcell death in the GCL of control retinas, and those treated for 30 minwith 25 μM of 18β-GA (B), or 50 μM of MFA (D) prior exposing to NMDA(300 μM). In experiments illustrated on upper panels, live and deadcells are marked with calcein-AM (green) and ethidium homodimer (EthD,red), respectively. In experiments on lower panels, retrograde labelingthrough optic nerve cut with LY was used to label RGCs and then retinasprocessed with EthD to detect dead cells. (E), Histogram summarizes theprotective effect of GJ blockers on RGCs against NMDA-inducedexcitotoxicity. The number of dead cells was counted manually per unitarea from 5 different visual fields in the GCL of whole-mount retinaspretreated with 18β-GA (n=12/3), or MFA (n=42/5), and numbers werenormalized to one obtained in retinas exposed to NMDA alone (control,n=44/5). **p<0.001 vs. control. Scale bar=100 μm.

FIGS. 3A-3E. Blockade of GJs reduced retinal injury and cell deathfollowing ischemia-reperfusion. (A) In control retinas, glial fibrillaryacidic protein (GFAP) expression was confined exclusively to astrocytesin the GCL and nerve fiber layer (NFL). (B) Seven days after reperfusionthe inner retinal thickness was reduced and the GFAP immunoreactivityappeared to traverse throughout the retinal layers and in the Müllercell processes. (C), Intravitreal injection of MFA (500 μM, 2 μl)prevented changes in the retinal morphology and reduced GFAPimmunoreactivity. (D) Histogram shows the nuclear counts in the GCL ofvertical sections of control (n=19/5; sections/retinas), and ischemicretinas in the presence (n=16/5) or absence (n=16/5) of MFA. (E)Histogram quantifies the GFAP immunofluorescence intensity throughoutthe vertical section in control and ischemic retinas with or without MFAtreatment. MFA was injected intravitreally either once 30 min before(blue, n=16/5), or twice at 3 and 24 hours after (yellow, n=12/3)ischemic insult vs. untreated ischemic retina). Retinal sections werecounterstained with PI. *p<0.01. Scale bar=50 μm.

FIGS. 4A-4G. Visualization of AC subpopulations with low and highdegrees of coupling to retrograde labeled RGCs. (A, C), Retinal GCs incontrol eyes were retrograde labeled for 40 min with NB, and doublelabeled with antibodies against amacrine cell marker calretinin (CR), orcholine acetyltransferase (ChAT). Cells positive to both NB andcorresponding AC markers are shown by arrowheads. (B, D), Blockade ofGJs with MFA significantly reduced the number of Neurobiotin-labeled,DR-IR amacrine cells in the INL, but had little effect on ChAT-IR cellnumbers. (E), Histogram shows that while the total number of CR-IR cells(per 500 μm section) in the inner nuclear layer (INL) of MFA-treatedretinas was comparable to that in control (n=18/4, p>0.1), the number ofthose labeled with Neurobiotin was significantly reduced byMFA-treatment (n=8/3). (F), There was no significant difference in thenumber of NB-positive ChAT-IR cells in control (n=16/3), and MFA-treatedretinas (n=6/3, p>0.1). (G), Histogram quantifying the percentage ofCR-IR and ChAT-IR amacrine cells in the INL that are coupled to RGCsunder control conditions. *p<0.01. Scale bars=50 μm.

FIGS. 5A-5L. Amacrine cells that are extensively coupled to RGCs showhigher susceptibility to NMDA-induced excitotoxicity. (A), Calretinin(CR) labels a large number of cells in the INL and GCL of controlretina. (B), In retinas exposed for 1 h to NMDA (300 μM), the number ofCR-positive cells was significantly reduced. (C), In vitro treatment ofretinas with MFA (50 μM, 30 min) prevented reduction in the number ofCR-IR cells in the INL. (D), Histogram summarizing NMDA-induced changesin the number of CR-positive ACs in the INL of untreated (n=18/3) andMFA-treated retinas (n=10/3). (E), Calbindin (CB) labeled horizontalcells in control retina. (F), Exposure to NMDA reduced the number ofCB-IR cells in the proximal INL. (G), Blockade of GJs with MFA preventedsuch reduction. (H), Histogram summarizing NMDA-induced changes in thenumber of CB-positive ACs in the INL of untreated (n=22/3) and MFAtreated retinas (n=6/3). The number of ChAT-IR ACs in control retinas(I-L), No significant change in the number of ChAT-IR ACs was observedin NMDA-treated retinas compared to controls (n=20/4). Retinal sectionswere counterstained with PI. *p<0.01 vs. control. Scale bars=50 μm.

FIGS. 6A-6L. Amacrine cells with a high degree of coupling to RGCs showhigher susceptibility to ischemia-reperfusion injury. (A-C),Calretinin-IR cells in control retinas and in those subjected toischemia-reperfusion injury in the absence and presence of MFA. (D),Histogram summarizing alteration in the number of CR-IR ACs in the INLof ischemic retinas with (n=26/5) or without (n=48/5) MFA-treatment.(E), Calbindin (CB) immunoreactivity was localized to the horizontalcells and ACs in the INL and sparse GCs. Three strata in the IPL werelabeled as well. (F), Changes in retinal morphology and the number ofCB-IR cells following ischemia/reperfusion. (G), Blockade of GJs by MFAlargely prevented a reduction in the number of CB-IR cells. (H),Histogram summarizing changes in CB-IR cells in the INL of control andischemic retinas untreated (n=17/3), or treated (n=21/3) with MFA.(I-L), No detectable change in the number of ChAT-IR ACs was observed inischemic retinas compared to levels in controls (n=25/4 each, p>0.1).Retinal sections were counterstained with PI. *p<0.001 vs. control.Scale bars=50 μm.

FIGS. 7A-7E. Gap junctions formed by Cx36 mediate bystander cell deathunder excitotoxic conditions. (A-D), Neuronal death was measured by EthDstaining in whole-mount retinas of heterozygous (Het), Cx36^(−/−),Cx36^(−/−)/45^(−/−) double knock-out (dKO), and Cx45^(−/−) mice. (E),Histogram quantifying excitotoxic cell death in the GCL of retinas fromHet and connexin knock out (KO) mice. Compared to Hets (n=35/3), thereduction in NMDA-induced cell death was statistically significant inretinas of Cx36^(−/−) (n=35/3), and Cx36^(−/−)/Cx45^(−/−) dKO (n=13/3)mice, but not in Cx45^(−/−) (n=17/3; p>0.1). *p<0.001 vs. Het. Scalebar=50 μm.

FIGS. 8A-8J. Gap junctions formed by Cx45 mediate bystander cell deathunder ischemic conditions. (A), lucifer yellow (LY) retrogradely labeledRGC somas and their axons in control retina of Het mouse, ischemic Hetretina (B), Cx36^(−/−) retina (C), and Cx45^(−/−) retina (D). (E),Immunoreactivity of GFAP in vertical sections of control retina andischemic retinas of Het (F), Cx36^(−/−) (G), and Cx45^(−/−) (H) mice.Following ischemia-reperfusion, the GFAP immunoreactivity wasupregulated throughout the retinal layers of Het and Cx36^(−/−), but notCx45^(−/−) mice. (I), Histogram shows the nuclear cell count (per 500 μmlength of vertical sections) in the GCL of control (n=54/5), andischemic retinas of Het (n=28/3), Cx36^(−/−) (n=24/4) and Cx45^(−/−)(n=74/5) mice. (J), Quantification of GFAP immunofluorescence intensityin the ischemic retinas of Het (n=24/4), Cx36^(−/−) (n=24/4), andCx45^(−/−) (n=22/4, p>0.1) mice. *p<0.001 vs. control. Scale bars=100 μm(A-D), 50 μm (E-H).

FIGS. 9A-9L. Changes in the immunolabeling of Cx36 and Cx45 in the IPLof retinas subjected to excitotoxic or ischemic insults. (A), In controlretina Cx36 immunoreactivity appeared as punctate labeling predominantlyin the inner half of the IPL. (B), In ischemic retinas the punctuatepattern of Cx36 labeling appeared as large clusters surrounding the cellnucleus. (C), NMDA-induced excitotoxicity had little effect on thepattern of Cx36 immunolabeling. (D), Histogram shows significantreduction in the intensity of punctate Cx36 immunoreactivity in the IPLof ischemic retinas (n=7/3) compared to that in controls (n=21/3). Nodetectable change in the Cx36 immunolabeling was observed underexcitotoxic conditions (n=8/3). (E), There was no significant differencein NMDA-induced cell death in the Het and Cx36^(−/−) retinas (n=21/3;p>0.1). (F), NMDA-induced cell death was significantly less inCx36^(−/−) (n=35/3) compared to Het retina (n=14/3). (G), Punctateimmunolabeling for Cx45 was observed throughout the IPL of controlretina. (H), The punctuate pattern and the intensity Cx45 labeling werenot altered by ischemia-reperfusion. I, NMDA markedly reduced Cx45immunolabeling. (J), Histogram quantifies Cx45 immunoreactivity in theIPL of retinas exposed to NMDA (n=8/4), compared to those in control(n=28/4), and ischemic retinas (n=26/4). (K), Cell death wassignificantly less in ischemic retinas of Cx45^(−/−) mice (n=24/5) ascompared to that in Het mice (n=30/5). (L), There was no statisticallysignificant difference in NMDA-induced cell death between Het andCx45^(−/−) retinas (n=18/3, p>0.1). **p<0.001. *p<0.01. Scale bars=20μm.

FIGS. 10A-10D. Elevation of intraocular pressure (IOP) and progressivecell loss in an experimental model of glaucoma induced by microbeadinjection into the eye. (A), Mean IOP measured over an 8 week periodfollowing injection of microbeads (red) or control phosphate bufferedsaline (PBS) (black) into eyes of wild type (WT) mice. The elevated IOPproduced by microbead injection started to decline at 2 weeks and so asecond injection was performed at 4 weeks following initial procedure.(B), Expression of GFAP in control retinas was limited mainly toastrocytes in the GCL and nerve fiber layer (NFL). Calibration bar forB=50 μm and pertains to C as well. (C), Eight weeks after microbeadinjection, the GFAP expression expanded to Müller cell processes thatextended vertically through the retinal layers. (D), Normalized cellcounts in the GCL (per 630 μm length) of retinal sections made undercontrol conditions and 1, 4 and 8 weeks after first microbead injection.There was a 20% decrease in total cell count in the GCL at 4 weeks and a36% decrease at 8 weeks after microbead injection compared to controlvalues. Bars represent mean±SD. **p<0.01; ***p<0.001 (Student's t-test).

FIGS. 11A-11F. Blockade of gap junctions with MFA prevents loss of cellsin GCL in microbead-injected eyes. Pharmacological blockade of GJs withMFA promotes RGC protection in experimental glaucoma. (A), Verticalsections of retinas from WT mice under control conditions and thosesubjected to microbead injection without (B), or with (C) intravitrealinjection of MFA. Under control conditions or 8 weeks after the initialmicrobead injection, eyes were retrogradely labeled with Neurobiotin(red), to visualize RGCs and the ACs and dACs to which they are coupledvia GJs. Sections were then subsequently immunolabeled for Brn3a(green), to identify RGCs, and counter-stained with the nuclearfluorogen 4′,6′-diamidino-2-phenylindole (DAPI) (blue), to visualizenuclei of all cells for cell counts. D-E. Histograms comparing the totalcell (RGC+dAC) and RGC counts in the GCL of microbead-injected retinasfrom WT mice untreated (D) or treated with MFA (E) to block GJs.Microbead injection clearly induced cell loss in the GCL, but blockadeof GJs prevented the loss. (F), Brn3A immunolabeling of RGCs in flatmount view. Calibration bar in A=50 μm and pertains to B and C. Cellcounts are per 1.3 mm length of retinal section. Bars representmean±standard error of the mean (SEM). **p<0.01 (Student's t-test).

FIGS. 12A-12I. Experimental glaucoma results in cell loss with ACpopulations. Immunolabels for AC populations, including CR, ChAT, andGABA were made in retinas of WT mice under control conditions (A, D, G)and 8 weeks after initial microbead injection (B, E, H). Cell countswere carried out in both the INL for ACs and GCL for dACs in verticalsections (C, F, I). There was a significant reduction of cells withinall the different populations of ACs/dACs after microbead injection.Cells counts are per 630 μm length of retinal section. Calibration barin A=50 μm and pertains to B-H. Histogram bars represent mean±SEM.*p<0.05, **p<0.01 (Student's t-test).

FIGS. 13A-13B. Ablation of Cx45 prevents loss of coupled dACs inexperimental glaucoma. Cell counts of total cells in the GCL based onDAPI label and coupled and uncoupled dACs taken as those labeled byretrograde Neurobiotin injection and those not labeled by Neurobiotinnor Brn3a, respectively. Counts were made in WT (A) and Cx45^(−/−) mice(B) under control conditions and 8 weeks after initial microbeadinjection. Microbead injection resulted in a loss of coupled dACs, butthere was a small increase in uncoupled dACs. However, there was no lossin coupled or uncoupled dACs in the Cx45^(−/−) mouse retinas. Barsrepresent mean±SEM. Differences in cell count values between control andmicrobead-injected WT mice were all significant (p<0.05; Studentst-test). None of the differences in cell count values between controland microbead-in-jected Cx45−/− mice were significant (p>0.1 for each,Students t-test).

FIGS. 14A-14E. Genetic ablation of Cx36, Cx45 or both Cx36/45significantly reduces cell loss in experimental glaucoma. (A),Normalized (to control) total cell counts in the GCL of WT under controlconditions and WT and connexin KO mice retinas 8 weeks after initialmicrobead injection to elevate IOP. There was a decrease in cell loss of45% and 50%, respectively, in Cx36^(−/−) and Cx45^(−/−) glaucomatousmouse retinas compared to values in glaucomatous WT retinas. We observeda 94% reduction in cell loss in glaucomatous Cx36^(−/−)/Cx45^(−/−)retinas indicating an additive effect of ablating both connexins. (B-C),Confocal images of vertical sections immunolabeled with Brn3aillustrating a significant protection of RGCs in microbead-injectedretinas of Cx36^(−/−)/Cx45^(−/−) mouse retina compared to WT.Calibration bar in B=50 μm and pertains to C as well. (D-E), Histogramsshowing changes in the number of total cells and RGCs in the GCL in WTand Cx36^(−/−)/Cx45^(−/−) mice under control conditions and 8 weeksafter initial microbead injection. Total cells counts were based onmeasures of DAPI-positive nuclei in the GCL. Values represent mean±SEM**p<0.01 (Student's t-test).

FIGS. 15A-15E. Injury-induced GFAP expression in the retina followingmicrobead injection is significantly reduced by deletion of Cx36 andCx45. (A), Before microbead injection, GFAP expression in WT mouseretinas is restricted to astrocytes and end feet of the Müller cells atthe inner limiting membrane. (B), Eight weeks after microbead injectionthere is an upregulation of GFAP in Müller cell processes that span theretinal layers. (C), This increase in GFAP expression was not seen inCx36^(−/−)/Cx45^(−/−) retinas 8 weeks after initial microbead injectionindicating a decrease in retinal injury. (D-E), Curves quantifying theexpression of GFAP in the retinas of the WT and KO mice under controland microbead-injected conditions. The profiles show a clear reductionin GFAP in the IPL of KO mice as compared to values in the WT afterinduction of experimental glaucoma. Scale bar in A=50 μm, and pertainsto B and C as well. Values represent mean±SEM **p<0.01 (Student'st-test).

DETAILED DESCRIPTION OF THE DISCLOSURE

Disclosed herein are methods for treating a condition of the retina byadministration of an inhibitor of connexin-36 and/or an inhibitor ofconnexin-45 to a subject who could benefit from the administrationthereof.

The disclosure provides results of a comprehensive study of the role ofgap junctions (GJs) in secondary neuronal death in the retina initiatedby excitotoxic or ischemic conditions. Significant numbers of retinalganglion cells are lost followed by subsequent loss of coupled amacrinecells after being subjected to excitotoxic or ischemic conditions.However, in accordance with the present disclosure, pharmacologicalblockade of gap junctions or genetic deletion of connexins increasedsurvivability of neurons by up to about 90%. The disclosure providesmethods of targeting specific connexins to reduce progressive cell lossinitiated by diverse neurodegenerative conditions. Moreover, the presentdisclosure reveals that apoptosis in a single cell can spread toneighboring cells via functional gap junctions and furthermore that gapjunctions mediate secondary cell-death in a connexin-specific manner.Therefore, in accordance with the present disclosure, it has beendetermined that gap junctions represent a novel, important target forneuroprotection.

The inventors have investigated the hypothesis that GJ-mediatedsecondary cell death forms a principle mechanism for the progressiveloss of cells under experimental glaucoma. The inventors have discoveredthat secondary or “bystander” cell death via GJs play a critical role inthe progressive loss of retinal ganglion cells (RGCs) and amacrine cells(ACs) in retinal conditions such as glaucoma. This disclosure revealsthe unexpected vulnerability of RGCs and ACs in glaucoma, the role of GJcoupling in the progression of cell loss, and which GJs, based on theirconnexin make-up, can be targeted to protect cells. This disclosure thusdefines a new mechanism for retinal bystander cell death, and thusprovides novel therapeutic targets for neuroprotection to preserve cellhealth and visual function in glaucomatous retinas. This level ofanalysis of the bystander effect has not been previously performed atany CNS locus. The retina, due to its GJ diversity, offers a uniquevenue to study this mechanism. Provided are treatments for manyneurodegenerative diseases of the retina, such as glaucoma, retinitispigmentosa and ischemic retinopathy, as well as those associated withother CNS loci.

Connexins, or gap junction proteins, are a family of structurallyrelated transmembrane proteins that assemble to form vertebrate gapjunctions. Connexins are four-pass transmembrane proteins withcytoplasmic C- and N-termini, a cytoplasmic loop (CL) and twoextra-cellular loops, (EL-1) and (EL-2). Connexins assemble into groupsof six to form hemichannels, or connexons, and two hemichannels thencombine to form a gap junction. The connexin gene family is diverse,with twenty-one identified members in the sequenced human genome, andtwenty in the mouse. The various connexins have been observed to combineinto both homomeric and heteromeric gap junctions, each of which mayexhibit different functional properties including pore conductance, sizeselectivity, charge selectivity, voltage gating, and chemical gating,depending at least in part on the combination of connexins present inthe GJ.

Connexin 36 (Cx36), also known as gap junction protein delta 2, is aconnexin with the nucleic acid sequence of SEQ ID NO: 1. The nucleicacid and amino acid sequence of Cx36 are available at Genbank AccessionNo. NM_020660. Connexin 45 (Cx45), also known as gap junction protein,gamma 1, is a connexin with the nucleic acid sequence of SEQ ID NO: 2.The nucleic acid and amino acid sequence of Cx45 are available atGenbank Accession No. NM_005497.

Accordingly, disclosed herein are methods for treating a condition ofthe retina, by administering an inhibitor of connexin 36 and/or aninhibitor of connexin 45 to a subject with a retinal condition. In someembodiments, both an inhibitor of connexin 36 and an inhibitor ofconnexin 45 are administered. The condition of the retina can beselected, for example, from glaucoma, macular degeneration, retinitispigmentosa, diabetic retinopathy and retinal ischemia.

In any of the above methods, the inhibitor can be selected from anantisense molecule directed to connexin 36 mRNA, an antisense moleculedirected to connexin 45 mRNA, and combinations thereof. Preferredantisense molecule inhibitors are those that selectively bind the mRNAsequence of connexin 36 (SEQ ID NO: 1) or the mRNA sequence of connexin45 (SEQ ID NO: 2).

A “connexin inhibitor” is a molecule that modulates or down-regulatesone or more functions or activities of a connexin or a connexinhemichannel (connexon) comprising a connexin of interest. Connexininhibitors include, without limitation, antisense compounds (e.g.,antisense polynucleotides), RNA interference (RNAi) and smallinterfering RNA (siRNA) compounds, antibodies and binding fragmentsthereof, and peptides and polypeptides (including “peptidomimetics” andpeptide analogs). Preferred connexin inhibitors are inhibitors ofconnexins found in neural GJs. Most preferred are inhibitors of connexin36 and/or connexin 45. In one embodiment, the connexin inhibitors of thepresent invention can modulate or prevent the transport of molecules,particularly molecules that initiate cell death, into and out of neuralcells.

Certain connexin inhibitors provide downregulation of connexinexpression, for example, by downregulation of mRNA transcription ortranslation, or decrease or inhibit a connexin protein, a connexinhemichannel or neural signaling activity (Asazuma-Nakamura et al., ExpCell Res., 2009, 315: 1190-1199; Nakano et al., Invest Ophthalmol VisSci., 2007, 49: 93-104; Zhang et al., Oncogene, 2001, 20: 4138-4149).

Anti-connexin polynucleotides include connexin antisense polynucleotidesas well as polynucleotides having functionalities that enable them todownregulate connexin expression. Other suitable anti-connexinpolynucleotides include RNAi polynucleotides, siRNA polynucleotides, andshort hairpin RNA (shRNA) polynucleotides.

Synthesis of antisense polynucleotides and other anti-connexinpolynucleotides (RNAi, siRNA, and ribozyme polynucleotides as well aspolynucleotides having modified and mixed backbones) is known to thoseof skill in the art. See, e.g., Stein C. A. and Krieg A. M. (Eds.),Applied Antisense Oligonucleotide Technology, 1998 (Wiley-Hiss). Methodsof synthesizing antibodies and binding fragments as well as peptides andpolypeptides (including peptidomimetics and peptide analogs) are knownto those of skill in the art. See, e.g., Lihu Yang et al., Proc. Natl.Acad. Sci. U.S.A., 1; 95(18): 10836-10841 (Sep. 1, 1998); Harlow andLane (1988) “Antibodies: A Laboratory Manual” Cold Spring HarborPublications, New York; Harlow and Lane (1999) “Using Antibodies” ALaboratory Manual, Cold Spring Harbor Publications, New York.

In one example, the downregulation of connexin expression is enacted byuse of antisense polynucleotides (such as DNA or RNA polynucleotides),and more particularly upon the use of antisense oligodeoxynucleotides(ODN). These polynucleotides (e.g., ODN) target the connexin protein(s)to be downregulated. Typically, the polynucleotides are single-stranded,but may be double-stranded.

The antisense polynucleotide may inhibit transcription and/ortranslation of a connexin. Preferably, the polynucleotide is a specificinhibitor of transcription and/or translation from the connexin gene ormRNA, and does not inhibit transcription and/or translation from othergenes or mRNAs. The product may bind to the connexin gene or mRNA either5′ to the coding sequence; and/or within the coding sequence, and/or 3′to the coding sequence. Administration of such connexin-targetedantisense polynucleotides inhibits bystander cell death in the retina.

The antisense polynucleotide is generally antisense to a connexin mRNA.Such a polynucleotide may be capable of hybridizing to the connexinmRNA, and may thus inhibit the expression of connexin by interferingwith one or more aspects of connexin mRNA metabolism (includingtranscription, mRNA processing, mRNA transport from the nucleus,translation or mRNA degradation). The antisense polynucleotide typicallyhybridizes to the connexin mRNA to form a duplex that can cause directinhibition of translation and/or destabilization of the mRNA. Such aduplex may be susceptible to degradation by nucleases.

The antisense polynucleotide may hybridize to all or part of theconnexin mRNA. Typically, the antisense polynucleotide hybridizes to theribosome binding region or the coding region of the connexin mRNA. Thepolynucleotide may be complementary to all of or a region of theconnexin mRNA. For example, the polynucleotide may be the exactcomplement of all or a part of connexin mRNA. However, absolutecomplementarity is not required, and polynucleotides that havesufficient complementarity to form a duplex having a melting temperatureof greater than about 20° C., 30° C. or 40° C. under physiologicalconditions (that is, under standard cellular conditions of salt, pH,etc., such as 0.09-0.15 M sodium phosphate, pH 6.5-7.2) are particularlysuitable for use in the present invention.

Thus, the polynucleotide is typically a homologue of a sequencecomplementary to the mRNA. The polynucleotide may be a polynucleotidethat hybridizes to the connexin mRNA under conditions of medium to highstringency, such as 0.03 M sodium chloride and 0.03 M sodium citrate atfrom about 50° C. to about 60° C.

For certain aspects, suitable polynucleotides are typically from about 6to 40 nucleotides in length. Preferably, a polynucleotide may be fromabout 12 to about 35 nucleotides in length, or more preferably fromabout 18 to about 32 nucleotides in length. According to another aspect,the polynucleotide may be at least about 40, for example, at least about60 or at least about 80 nucleotides in length; and up to about 100,about 200, about 300, about 400, about 500, about 1,000, about 2,000 orabout 3,000 or more nucleotides in length.

In one preferred aspect, the antisense polynucleotides are targeted tothe mRNA of only one connexin protein. Most preferably, this connexinprotein is connexin 36 or 45. Polynucleotides targeted to connexins 36and 45 proteins may be used in combination. In addition, antisensenucleotides targeted to connexins 36 and 45, in combination withantisense nucleotides targeted to one or more additional connexinproteins (such as connexins 26, 30, 30.3, 31.1, 32, 37, 40, 43, 45, and47), are used. These are examples of human connexin proteins. In thecase of other animal species, mRNA of the corresponding connexin istargeted. In one embodiment, the combination of antisense nucleotidesdoes not include an antisense nucleotide targeted to connexin 43.

Individual antisense polynucleotides may be specific to connexin 36 or45, or may hybridize to connexin 36 and/or 45 and 1, 2, 3 or moreadditional connexins. Specific polynucleotides will generally targetsequences in the connexin gene or mRNA that are not conserved betweenconnexins, whereas non-specific polynucleotides will target conservedsequences for various connexins.

The antisense polynucleotides may be chemically modified. This mayenhance their resistance to nucleases and may enhance their ability toenter cells. For example, phosphorothioate oligonucleotides may be used.Other deoxynucleotide analogs include methylphosphonates,phosphoramidates, phosphorodithioates, N3′P5′-phosphoramidates andoligoribonucleotide phosphorothioates, and their 2′-O-alkyl analogs and2′-O-methylribonucleotide methylphosphonates. Alternatively, mixedbackbone oligonucleotides (“MBOs”) may be used. MBOs contain segments ofphosphorothioate oligodeoxynucleotides and appropriately placed segmentsof modified oligodeoxy- or oligoribonucleotides. MBOs have segments ofphosphorothioate linkages and segments of other modifiedoligonucleotides, such as methylphosphonate, which is non-ionic, andvery resistant to nucleases or 2′-O-alkyloligoribonucleotides. Methodsof preparing modified backbone and mixed backbone oligonucleotides areknown in the art.

Polynucleotides (including ODNs) directed to connexin proteins can beselected in terms of their nucleotide sequence by any suitable approach.For example, the computer programs MacVector and OligoTech (from OligosEtc., Eugene, Oreg., USA) can be used. Once selected, the ODNs can besynthesized using a DNA synthesizer.

For example, the polynucleotide may be a homologue of a complement to asequence of connexin 36 or connexin 45 mRNA. Such a polynucleotidetypically has at least about 70% homology, preferably at least about80%, at least about 90%, at least about 95%, at least about 97%, atleast about 98% or at least about 99% homology with a portion of SEQ IDNO: 1 or SEQ ID NO: 2, for example, over a region of more than at leastabout 15, at least about 20, at least about 40, or at least about 100contiguous nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2.

Homology may be calculated based on any method in the art. For example,the UWGCG Package provides the BESTFIT program, which can be used tocalculate homology (for example, used on its default settings) (Devereuxet al. (1984) Nucleic Acids Research 12, p 387-395). The PILEUP andbasic local alignment search tool (BLAST) algorithms can be used tocalculate homology or line up sequences (typically on their defaultsettings), for example, as described in Altschul, S. F. (1993) J MolEvol 36: 290-300; Altschul, S. F. et al. (1990) J Mol Biol 215: 403-10.

Software for performing BLAST analyses is publicly available onlinethrough the National Center for Biotechnology Information. Thisalgorithm involves first identifying high-scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence that eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul, S. F. (1993) J MolEvol 36: 290-300; Altschul, S. F. et al. (1990) J Mol Biol 215: 403-10).These initial neighborhood word hits act as seeds for initiatingsearches to find HSPs containing them. The word hits are extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a word length (W), the BLOSUM62 scoring matrix(Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci USA 89:10915-10919), alignments (B) of 50, expectation (E) of 10, M=5, N=4, anda comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences. See, e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence to a second sequence isless than about 1, preferably less than about 0.1, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The homologous sequence typically differs from the relevant sequence byat least about 2, 5, 10, 15, 20 or more mutations (which may besubstitutions, deletions or insertions). These mutations may be measuredacross any of the regions mentioned above in relation to calculatinghomology. The homologous sequence typically hybridizes selectively tothe original sequence at a level significantly above background.Selective hybridization is typically achieved using conditions of mediumto high stringency (for example, 0.03M sodium chloride and 0.03 M sodiumcitrate at from, about 50° C. to about 60° C.). However, suchhybridization may be carried out under any suitable conditions known inthe art (see Sambrook et al. (1989), Molecular Cloning: A LaboratoryManual). For example, if high stringency is required, suitableconditions include 0.2×SSC at 60° C. If lower stringency is required,suitable conditions include 2×SSC at 60° C.

Alternatively, the inhibitor can be a small molecule inhibitor or aconnexin binding protein (including peptides, peptidomimetics,antibodies, antibody fragments, and the like).

The term “small molecule” as used herein refers to molecules thatexhibit a molecular weight of less than 5000 Da, more preferred lessthan 2000 Da, even more preferred less than 1000 Da and most preferredless than 500 Da. Compounds or competitor compounds that are syntheticand/or naturally occurring “small molecule” compounds, e.g. drugs,metabolites, prodrugs, potential drugs, potential metabolites, potentialprodrugs and the like, are preferred for use in the methods describedand claimed herein. Preferably, said compounds or competitor compoundsare selected from the group consisting of synthetic or naturallyoccurring chemical compounds or organic synthetic drugs, more preferablysmall molecules, organic drugs or natural small molecule compounds.Exemplary small molecule inhibitors include 18-Beta-glycyrrhetinic acid(18Beta-GA) and meclofenamic acid (MFA). Small molecule inhibitors maybe screened from small molecule libraries available in the art forability to inhibit connexin 36 and/or connexin 45.

Further disclosed herein are use of inhibitors of connexin 36 and/orconnexin 45, including, but not limited to, antisense polynucleotides,RNAi and siRNA compounds, antibodies and binding fragments thereof,peptides and polypeptides, and small molecules, in the preparation of apharmaceutical composition for the treatment of retinal conditions, suchas glaucoma, macular degeneration, retinitis pigmentosa, diabeticretinopathy and retinal ischemia. Additionally disclosed are methods oftreatment for retinal conditions, involving administration of inhibitorsof connexin 36 and/or connexin 45.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the disease course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Therapeutic effects of treatment includewithout limitation, preventing occurrence or recurrence of disease,alleviation of symptoms, diminishment of any direct or indirectpathological consequences of the disease, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis.

Any of the above methods can include repeat administration of theinhibitor or inhibitors for a period of 1 week to 1 year, such as 1-4weeks, 1-3 months, 1-6 months, 6 months to 1 year, or a year or more.Administration can be, for example, one, two, or three times daily, onceevery 2-3 days, or once per week. Particularly contemplated is topicaladministration, such as a drop to be administered to the eye. Topicaladministration includes directly applying, laying, or spreading on oraround the eye, e.g., by use of an applicator such as a wipe, a contactlens, a dropper, or a spray.

Also disclosed herein are pharmaceutical compositions for treatment of aretinal condition. The compositions include an inhibitor of connexin 36and/or an inhibitor of connexin 45. The inhibitor or inhibitors can beselected from an antisense molecule directed to connexin 36 mRNA, anantisense molecule directed to connexin 45 mRNA, and combinationsthereof. In another example, the composition includes a small moleculeinhibitor of connexin 36 and/or a small molecule inhibitor of connexin45. In a further example, the inhibitor or inhibitors can be selectedfrom 18-Beta-glycyrrhetinic acid (18Beta-GA) and meclofenamic acid(MFA). Any of the disclosed compositions can be formulated for topicaladministration or intraocular injection to the eye.

Particularly contemplated are compositions that include an inhibitor ofconnexin 36 and/or an inhibitor of connexin 45 in a pharmaceuticallyacceptable carrier. As used herein, the phrase “pharmaceuticallyacceptable” means the carrier, or vehicle, does not cause an adversereaction when administered to a mammal. Such carriers are non-toxic anddo not create an inflammatory or anergic response in the body.Pharmaceutically acceptable carriers for practicing the inventioninclude well known components such as, for example, culture media andphosphate buffered saline. Additional pharmaceutically acceptablecarriers and their formulations are well-known and generally describedin, for example, Remington's Pharmaceutical Science (18th Ed., ed.Gennaro, Mack Publishing Co., Easton, Pa., 1990) and the Handbook ofPharmaceutical Excipients (4th ed., Ed. Rowe et al. PharmaceuticalPress, Washington, D.C.), each of which is incorporated by reference.

A composition containing an inhibitor of connexin 36 and/or an inhibitorof connexin 45 can be formulated for topical administration. Forms ofthe composition include, but are not limited to, solutions, ointments,gels, emulsions, suspensions, gel shields, and the like. In oneembodiment, the composition is formulated as an aqueous-based creamexcipient, which can be applied to the eye at bedtime, but may also beapplied any time throughout the day. In another embodiment, thecomposition is formulated as a solution or suspension and is appliedtopically in the form of eye drops. Any solution suitable for topicalapplication in which a disclosed inhibitor is soluble can be used; e.g.,sterile water, Sorenson's phosphate buffer, and the like.

In other embodiments, a composition is formulated to have propertiessuch as sustained-release or improved stability. For example, apolymeric matrix composition containing an inhibitor of connexin 36and/or an inhibitor of connexin 45 can be topically applied to the eyeto achieve sustained release.

Compositions containing an inhibitor of connexin 36 and/or an inhibitorof connexin 45 can include additional ingredients, additives or carriersuitable for use in contact on or around the eye without undue toxicity,incompatibility, instability, irritation, allergic response, and thelike. Additives such as solvents, bases, solution adjuvants, suspendingagents, thickening agents, emulsifying agents, stabilizing agents,buffering agents, isotonicity adjusting agents, soothing agents,preservatives, corrigents, olfactory agents, coloring agents,excipients, binding agents, lubricants, surfactants,absorption-promoting agents, dispersing agents, preservatives,solubilizing agents, and the like, can be added to a formulation whereappropriate.

The compositions of the present invention can include other activeagents for treatment of retinal conditions, including, but not limitingto, anti-infective agents, antibiotics, antiviral agents,anti-inflammatory drugs, anti-allergic agents including anti-histamines,vasoconstrictors, vasodilators, local anesthetics, analgesics,intraocular pressure-lowering agents, immunoregulators, anti-oxidants,vitamins and minerals, proteases and peptidases that breakdownendogenous opioids, and the like.

Further disclosed herein are uses of any of the disclosed compositionsfor the treatment of a condition of the retina. In some examples, thecondition of the retina can be selected from glaucoma, maculardegeneration, retinitis pigmentosa, diabetic retinopathy and retinalischemia.

The present disclosure is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1. Materials and Methods

Animals:

Experiments were performed on: (1) wild type (WT) C57BL/6 mice; (2)connexin knockout (KO) mice Cx36^(−/−), Cx45^(−/−), andCx36^(−/−)/45^(−/−) (double KO) and their heterozygous (Het)littermates; (3) the hereditary glaucoma model DBA/2J mouse strain; and(4) transgenic CB2, Grik4, and Kokcng (Kcng4, Potassium voltage-gatedchannel subfamily G member 4)-cre lines in which select RGC and (d)ACsubtypes express fluorescent markers and can be visualized forhistological or electrophysiologic experiments. All strains arecurrently maintained by our lab in the SUNY College of Optometry animalfacility. The Cx36−/− mice and littermates were derived from F2C57BL/6-129SvEv mixed background litters (Kameritsch P, et al., CellDeath Dis. 4:e584(2013)). The Cx45−/− were generated by crossingCx45fl/fl mice with mice expressing Cre recombinase under control of theneuron-directed nestin promoter to yield Cx45fl/fl:Nestin-cre mice(Blankenship A G, et al., J Neurosci. 31:9998-10008 (2011); Pang J J, etal., Invest Ophthalmol Vis Sci. 54:5151-5162. (2013)). All experimentswere performed in adult mice of either sex.

Retina-Eyecup Preparation.

All animal procedures were in compliance with the NIH Guide for the Careand Use of Laboratory Animals and approved by the Institutional AnimalCare and Use Committees at SUNY College of Optometry. Experiments wereperformed on retinas of wild type (WT), connexin knockout (KO) mice(Cx36^(−/−), Cx45^(−/−), and Cx36^(−/−)/45^(−/−) dKO), and theirheterozygous (Het) littermates. The methods used to impale and labelneurons have been described previously (Hu et al., J Neurosci23:6768-2777 (2003); Volgyi et al., Journal of Comparative Neurology512:664-687 (2009)). Briefly, flattened retina-eyecups were placed in asuperfusion chamber, which was mounted on the stage of a BX51WI lightmicroscope (Olympus) within a light-tight Faraday cage. An IR-sensitiveCCD camera (Dage) captured the retinal image, which was displayed on avideo monitor. The retina-eyecups were superfused with a modified,oxygenated Ames medium maintained at 35° C.

Intracellular Injections.

For intracellular injections, neurons were visualized and impaled withstandard, sharp glass microelectrodes filled with cytochrome C (CytC, 10mg/ml) and Neurobiotin (4%) in 0.1 M Tris buffer. Substances wereinjected iontophoretically for 15-20 min using a sinusoidal current (3Hz, 1-5 nA p-p).

Microbead Injection.

Experimental glaucoma in mice is induced by IOP elevation achieved byintracameral injections of 10 μm polystyrene microbeads (Invitrogen) aspreviously described (Chen H, et al., Invest Ophthalmol Vis Sci.52:36-44 (2011)). The intracameral injections are made unilaterally with2 μl of microbead suspension containing ˜7.2×106 beads using a glassmicropipette attached to a microliter syringe. An equivalent volume ofphosphate-buffered saline (PBS) is injected in contralateral eyes toserve as a control. Measurements of IOP are made with thecommercially-available Tonolab tonometer (Colonial Medical Supply) andare performed weekly for up to 8 weeks after the microbeads injection.Measurements are made between 10 AM and 12 PM, to minimize the effect ofdiurnal IOP variations. Six IOP measurements are made at each intervaland averaged.

Immunocytochemistry.

After experimental treatment, retinas were fixed with 4%paraformaldehyde in a 0.1 M phosphate buffer solution (PBS; pH 7.4) for30 minutes at room temperature, cryoprotected in 30% sucrose, embeddedin Tissue-Tek OCT Compound (Andwin Scientific) and frozen. Cryosections(18-20 μm thick) were cut and mounted on microscope slides. Forimmunostaining, sections were blocked in 3% donkey serum in 0.1M PBSsupplemented with 0.5% Triton X-100, and 0.1% NaN3 for 1 h at roomtemperature. Primary antibodies were diluted in 0.1M PBS with 0.5%Triton X-100, 0.1% NaN3 and 1% donkey or goat serum. Tissues were thenincubated with primary antibodies for 3 hours at room temperature orovernight at 4° C. The following primary antibodies were used: rabbitanti-calretinin 1:2000, rabbit anti-calbindin 1:1000, goat anti-ChAT1:100 (all from Millipore), rabbit anti-GFAP 1:1000 (Invitrogen), rabbitanti-active caspase 3 1:200 (Abcam); mouse anti-Cx35/36 1:300, mouseanti-Cx45 1:300 (both from Millipore). After extensive washing with 0.1MPBS, tissues were incubated for 1 hour in secondaryanti-goat/rabbit/mouse antibodies conjugated to Alexa-488 or Alexa-594(1:200, Molecular Probes). Retinal sections were counterstained with thenuclear dye propidium iodide (PI, Molecular Probes). Neurobiotin wasvisualized with Alexa-488/594 conjugated streptavidin (Molecular Probes,1:200). Tissues were then flat-mounted in Vectashield media (VectorLabs) and fluorescent images were taken using a fluorescent microscopeor an Olympus FV1200 MPE confocal microscope.

Induction of Cell Death.

Various methods were employed to produce cell death. (1) Single cellapoptosis: CytC was injected into individual RGCs or Müller cells for 15minutes after which retinas were incubated for 4 hours in oxygenatedAmes medium before fixation. After streptavidin histology, apoptoticcells were detected with an anti-active caspase 3 antibody or withTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)staining (2) Excitotoxicity: To assess the contribution of GJs to celldeath within populations of RGCs and amacrine cells we conductedparallel experiments in which retinas were preincubated for 20 minutesin normal Ames medium or in one containing the GJ blockers meclofenamicacid (MFA; 50 μM) or 18-beta-glycyrrhetinic acid (18Beta-GA; 25 μM).Both control and drug-treated retinas were then exposed for 1 hour toNMDA (100-300 μM) to induce excitotoxicity followed by 4 hours in thecontrol Ames solution. (3) Retinal ischemia: Transient in vivo retinalischemia was induced by introducing into the anterior chamber a 33-gaugeneedle attached to a saline-filled reservoir (0.9% sodium chloride) thatwas raised above the animal so as to increase intraocular pressure (IOP)to a level 120 mm Hg above systolic blood pressure. MFA (2 μl, 500 μM)was administered intravitreally either 30 minutes before or 3 and 24hours following the ischemic insult. The opposite eye was cannulated,but maintained at normal IOP to serve as a normotensive control. After40-50 minutes, the needle was withdrawn and ischemia was evidenced bycorneal whitening. After 7 days of reperfusion, mice were sacrificed andeyes were processed to assess retinal damage and neuronal death. We alsoemployed oxygen-glucose deprivation (OGD) to induce in vitro ischemia,in which retina-eyecups or isolated retinal whole-mounts were exposedcontinuously to either a control, oxygenated Ringer solution, or onethat was glucose-free and deoxygenated by bubbling extensively with 95%N₂/5% CO₂ at 34° C. Following 60 minutes in the OGD environment, retinaswere transferred to a control, oxygenated Ames medium for 4 hours beforeprocessing to evaluate cell death.

Retrograde Labeling and Visualization of Coupled Cells.

We used retrograde labeling of RGCs with Neurobiotin to visualizepopulations of amacrine cells in the inner nuclear layer (INL) to whichthey were coupled. Globes with attached optic nerves were submersed inoxygenated Ames medium and a drop of Neurobiotin (4% in 0.1 M Trisbuffer) was applied to the cut optic nerve for 40 minutes. Forretrograde labeling limited to RGCs, Neurobiotin was replaced withLucifer yellow (LY, 3%). In separate experiments, retrograde labelingwas performed in both eyes prior to incubating one eye in Ames mediumcontaining MFA (50 μM for 30 minutes) after which both eyes were exposedto NMDA (300 μM) for 1 hour. Frozen retinal sections (20 μm thick) werecut on a cryostat and processed for assessment of cell death in theinner nuclear layer (INL) and ganglion cell layer (GCL).

Assessment of Cell Injury and Cell Death.

Apoptotic and necrotic cell death were assessed by cell countsfollowing: (1) staining with Live/Death Viability Assay (calceinAM/ethidium homodimer (EthD) (Invitrogen); (2) TUNEL, or; (3) labelingfor activated caspase 3. For population studies, dead cells were countedmanually within 500×500 μm areas (5 areas per retina) from micrographsof whole-mounts using images acquired by confocal microscope. Nuclearcell counts were made per unit length (500 μm) of frozen retinalcross-sections labeled with propidium iodide (2 μg/ml).

In some experiments, cells counts were limited to retrograde labeledRGCs in the GCL or certain subpopulation of amacrine cells, identifiedby specific markers, such as calretinin (CR), calbindin (CB) and cholineacetyltransferase (ChAT) in the INL. Fluorescence intensity measureswere made of glial fibrillary acidic protein (GFAP), which isoverexpressed in Müller glial cells following retinal injury. Expressionof GFAP and connexins were quantified by analysis of confocal imageswith Metavue software (Molecular Devices). Average pixel fluorescenceintensities were measured by using uniform rectangular areas (3-5 perimage) extending either from the GCL to the outer limiting membrane forGFAP or through the IPL for connexins. The intensity values were thenaveraged for at least 5 images from 3-5 independent experiments and datawere normalized to controls.

Microbead-Induced Glaucoma.

Experimental glaucoma was induced by intracameral injections ofpolystyrene microbeads (Chen H, et al., Invest Ophthalmol Vis Sci.52:36-44 (2011)) in one eye of wild type (WT) mice, connexin knockout(KO) mice and their heterologous (HET) control littermates, with shaminjection of the second eye as control. IOP measurements were performedweekly and animals were sacrificed at 1, 4, or 8 weeks after initialbead injection. To access cell loss, RGCs and coupled ACs wereretrogradely labeled with optic nerve injection of Neurobiotin andvertical retinal sections were counterstained with DAPI to determineoverall nuclear counts. RGCs were also labeled by Brn3a antibody foridentification. In addition to cell counts, overall retinal damage wasassessed by GFAP expression. In some experiments gap junctions wereblocked with meclofenamic acid (MFA) intravitreal weekly injections orgenetically ablated in connexin KO mice (Cx36^(−/−), Cx45^(−/−), andCx36^(−/−)/Cx45^(−/−) mice).

Statistics.

Data are presented as mean±standard mean error. The number ofmeasurements carried out for a given experiment (n) are given as x/ywhere x is the total number of samples in which the measures were made(e.g., flat mount areas or sectional lengths) and y is number ofretinas. For microbead injection experiments, data are presented asmean±SEM per 1.3 mm length of vertical section. Statistical comparisonswere assessed using Student's t-test. Values of p≦0.05 are consideredstatistically significant.

Example 2. Gap Junctions Mediate Secondary Cell Death in the Retina

We initially examined whether GJ-mediated secondary cell death occurredin the retina. In these experiments, we performed intracellularinjections of single RGCs with CytC to stimulate apoptosis andNeurobiotin to assess GJ coupling. To determine whether apoptosis in asingle cell could spread to neighbors through a mechanism other thanGJs, we first examined RGCs that were not tracer coupled to other cells(FIG. 1A). We found that injection of CytC initiated cell death in theinjected cell within the experimental timeframe of 3-4 hours, but noother neuron neighbors were lost (FIG. 1B,C). In contrast, injection ofCytC into RGCs that were coupled to ganglion and/or amacrine cellsresulted in the death of not only the injected cell, but also thecoupled neighbors (FIG. 1D-F). These results confirmed that secondarycell death does occur in the retina and is dependent on functional GJs.It is important to note that CytC, at 12,000 Daltons, is far too large amolecule to pass across a GJ, indicating that other toxic molecules mustbe moving intercellularly to promote cell death in coupled neighbors. Wealso found that secondary cell death occurred in coupled Müller cells,indicating that this mechanism can result in progressive death of gliain addition to neurons (FIG. 1G-I).

Example 3. Pharmacological Blockage of Gap Junctions Reduces RGC DeathUnder Excitotoxic or Ischemic Conditions

To determine the contribution of secondary cell death to the loss ofRGCs produced under different neurodegenerative conditions, we examinedthe effect of GJ blockade on cell loss in the GCL. In initialexperiments, we induced excitotoxic cell death by incubatingretina-eyecups in 100-300 μM NMDA (FIG. 2A,C). Cell death counts werethen performed in these retinas (n=44/5) and compared to retinas thatwere incubated in the non-selective GJ blockers 18Beta-GA (25 μM) or MFA(50 μm) prior to exposure to excitotoxic conditions (FIG. 2B,D). Wefound that prior blockade of GJs with either 18Beta-GA (n=12/3) or MFA(n=42/5) significantly reduced (p<0.001 for both drugs) cell death inthe GCL induced by excitotoxicity (FIG. 2E). Overall, NMDA-induced celldeath was reduced dramatically in the GCL of mouse retinas pretreatedwith 18Beta-GA or MFA compared to those treated with NMDA alone. Incontrol experiments, application of 18Beta-GA or MFA alone did notaffect cell viability (p>0.1), supporting the notion that the GJblockers had no spurious toxic effects. In addition, MFA, at theconcentrations tested here, shows no inhibitory effect on NMDA receptorsin neurons, suggesting that the attenuation of NMDA-induced cell deathby GJ blockade was not due to reduced NMDA receptor activity. Overall,the significant protective effects of pharmacological GJ blockadesuggest that secondary cell death is responsible for the progressiveloss of the vast majority of RGCs under excitotoxic conditions.

In a second phase of experiments, we examined the role of secondary celldeath in RGCs loss associated with ischemic conditions of the retina. Weassessed retinal damage subsequent to ischemia induced in vivo byraising IOP above normal systolic levels. In control retinas (n=19/5),GFAP expression was confined exclusively to astrocytes in the GCL (FIG.3A,E). Expression of GFAP in ischemic retinas, however, wassignificantly upregulated as evidenced by the spread of immunolabelingto Müller cell processes at all retinal layers (p<0.01) (FIG. 3B, E).Ischemia also produced a significant reduction in nuclear counts in theGCL (p<0.01) accompanied by a marked thinning of the retina,particularly the inner layers (FIG. 3A,B,D).

To assess the role of GJ-mediated secondary cell damage in ischemicinjury of the retina, eyes were injected intravitreally with MFA eitherbefore or after insult. Treatment with MFA prior to ischemic inductionpreserved retinal thickness, cell counts in the GCL, and GFAP expressionto levels seen in control retinas (FIG. 3C,D,E). Moreover, theprotective effect of MFA administered 3 and 24 hours after transientischemia was evidenced by maintenance of normal levels of retinalthickness, GFAP expression, and cell counts in the GCL measured one weekafter insult; p<0.01 for all measures (FIG. 3C,D,E).

Example 4. Gap Junctions Mediate Amacrine Cell Loss Following RetinalInjury

15 of the 22 morphological subtypes of RGCs in the mouse retina arecoupled to amacrine cells. This extensive coupling suggests that theGJ-mediated secondary cell death may also progress from RGCs to coupledamacrine cell neighbors. To test this idea, we examined the loss ofamacrine cell populations immunolabeled with calretinin (CR), calbindin(CB) or choline acetyltransferase (ChAT).

Initial experiments were carried out to determine whether CR-, CB-, andChAT-immunopositive amacrine cells (ACs) were indeed coupled to RGCs.Ganglion cell somata were retrogradely labeled with Neurobiotin throughthe cut optic nerve. A large number of cells in the INL and GCL wereimmunoreactive to CR and ChAT with three distinct IPL bands ofCR-labeled dendritic processes and two clear ChAT bands of starburst-aand starburst-b ACs (FIG. 4A,C). In addition to CR-positive RGCs (ordisplaced amacrine cells) in the GCL, we found (n=18/4) thatapproximately one-half of the CR-positive, presumed ACs in the INLshowed Neurobiotin labeling (FIG. 4A, E, G), suggesting that they werecoupled to RGCs. Indeed, blockade of GJs with MFA prior to retrogradelabeling effectively eliminated all colocalized labeling of CR-positiveACs in the INL with Neurobiotin (n=8/3, p<0.01) (FIG. 4B,E).CB-immunoreactive labeling in the mouse retina is known to mimiclabeling for CR, suggesting that they may label the same subpopulationsof ACs and RGCs. Our results from CB-immunolabeled retinas were similarto those described for CR.

Although starburst ACs in the rabbit retina are not coupled to RGCs, wefound a small number (less than 10%) of ChAT-positive cells in the INLof the mouse retina that were labeled with Neurobiotin (n=16/3) (FIG.4C, F, G). However, application of MFA did not produce a significantreduction in the number of ChAT-positive ACs labeled with Neurobiotin(n=6/3; p>0.1) (FIG. 4D, F). We conclude that, at best, only a smallnumber of starburst-a ACs are coupled to RGCs.

In the next set of experiments, we examined how AC death due toexcitotoxicity was affected by blockade of GJs with MFA. Application ofNMDA produced a significant reduction of CR- (n=18/3; p<0.01) andCB-immunopositive (n=22/3; p<0.01) subpopulations of ACs in the INL(FIG. 5A-H). However, blockade of GJs with MFA prevented the loss of CR-(n=10/3) or CB-immunolabeled (n=6/3) ACs (p<0.01), preserving levelsseen in control retinas (FIG. 5C, D, G, H). In contrast, NMDA-inducedexcitoxicity did not produce a significant reduction in the number ofChAT-positive ACs in the INL (n=20/4; p>0.1), nor was this affected byGJ blockade with MFA (n=17/4; p>0.1) (FIG. 5L).

Amacrine cell death following transient ischemia paralleled the resultsseen following excitotoxic insult. Ischemia produced a significant lossof CR- (n=48/5; p<0.001) and CB-immunoreactive (n=17/3; p<0.001)subpopulations of ACs in the INL and a disorganization of the dendriticbands in the IPL (FIG. 6A-H). Application of MFA prevented the loss ofboth the CR- (n=26/5; p<0.001) and CB-immunoreactive (n=21/3; p<0.001)ACs, maintaining levels to that seen in control retinas (FIG. 6C, D, G,H). In contrast, induction of ischemia had no statistically significantimpact on the number of ChAT-immunopositive amacrine cells in the INL(n=25/4; p>0.1) (FIG. 6L). As expected, treatment of ischemic retinaswith MFA had no effect on the number of ChAT-positive ACs in the INL(n=23/4; p>0.1) (FIG. 6K, L).

Example 5. Gap Junctions Mediate Secondary Cell Death in aConnexin-Specific Manner

The inner plexiform layer (IPL) of the retina contains an assortment ofGJs formed between RGCs, ACs, and bipolar cell axon terminals thatexpress at least three different connexin subunits. This diversityraises the notion that different cohorts of GJs, possibly based on theirconnexin profiles, may be responsible for secondary cell death in theinner retina arising from different primary insults. Our results usingthe GJ blockers MFA and 18Beta-GA did not address this issue. To testthis idea, we therefore examined the extent of excitotoxic and ischemiccell death in mice in which Cx36 and/or Cx45, the two most highlyexpressed subtypes in the IPL, were genetically deleted.

We induced excitotoxic cell death as described above by application ofNMDA to retinas of Cx36^(−/−), Cx45^(−/−), and Cx36^(−/−)/Cx45^(−/−) dKOmouse retinas and their heterozygous littermates. Detection of deadcells in the GCL showed that NMDA-induced excitotoxic cell death wasmarkedly reduced in Cx36−/− mouse retinas (n=35/3) as compared to levelsfound in Het littermates (n=35/3) or WT mice (n=14/3; p<0.001 for both)(FIG. 7A, B,E). In contrast, the extent of cell death in the GCL ofCx45−/− mouse retinas following exposure to NMDA was not statisticallydifferent from control levels in Het or WT mice (n=17/3; p>0.1) (FIG.7A, C, E). We next induced excitotoxic cell death inCx36^(−/−)/Cx45^(−/−) dKO mouse retinas (n=13/3) and found that thelevel of cell death in the GCL was indistinguishable from those found inNMDA-treated retinas of Cx36^(−/−) mice (p>0.1) (FIG. 7B, D, E). Thus,the degree of cell death was not additive when both Cx36- andCx45-expressing gap junctions were deleted. Overall, these resultsindicated that whereas GJs expressing Cx36 played a role in secondarycell death associated with excitotoxicity, those expressing Cx45 made nosignificant contribution.

In the next phase of experiments, we induced transient retinal ischemiain vivo in Cx36^(−/−) and Cx45^(−/−) mice and their Het littermates.After 7 days of survival, evaluation of retrograde labeling of GCs withLucifer yellow (LY) in whole mount retinas revealed a significant lossof axonal processes in Cx36−/− and Het mouse retinas as compared tocontrol levels (FIG. 8A-C). In contrast, ischemic retinas fromCx45^(−/−) mice showed axonal labeling that was comparable to that seenin control retinas (FIG. 8A, D). Consistent with these findings,ischemia resulted in a marked reduction of cells in the GCL ofCx36^(−/−) (n=24/4) and Het littermate mouse retinas (n=28/3; p<0.001for both), but no significant loss in the GCL of Cx45^(−/−) mice(n=74/5), when compared to control levels (n=54/5; p>0.1) (FIG. 81). TheGFAP immunoreactivity was also markedly increased in Müller cellprocesses following ischemic insult of Cx36^(−/−) (n=24/4) and Het(n=24/4) mouse retinas (p<0.001), but showed levels in Cx45^(−/−) mouseretinas (n=22/4) that were indistinguishable from that measured incontrol retinas (p>0.1) (FIG. 8E-H, J).

A differential contribution of Cx36- and Cx45-expressing GJs tosecondary neuronal death was also observed under condition ofoxygen-glucose deprivation (OGD), an in vitro model of ischemia.Exposure to OGD conditions produced a significant loss of neurons in theGCL of Cx36^(−/−) (n=12/3) and Het mouse retinas (n=12/3; p<0.001), butproduced no significant loss of cells in the GCL of Cx45−/− mouseretinas (n=10/3; p>0.1).

We then investigated any changes in the distribution of Cx36- andCx45-expressing GJs under excitotoxic and ischemic insult that couldinstruct their differential roles in secondary cell death (FIG. 9E, F,K, L). Induction of ischemia produced a dramatic reduction in theexpression of Cx36 puncta in the IPL (n=7/3), compared to that incontrol retinas (n=21/3; p<0.001) (FIG. 9A, B). Instead, we foundintense Cx36 immunolabeling in RGC somata indicating an accumulation ofthe protein in the cytoplasm (FIG. 9B, inset). The expression of Cx45puncta in the IPL was unaffected by ischemic insult (n=26/4; p>0.1)(FIG. 9G, H, J). However, exposure of retinas to NMDA to induceexcitotoxicity had no effect on the expression of Cx36 puncta in the IPL(n=8/3; p>0.1), but dramatically reduced the expression of Cx45 (n=8/4),compared to control levels (n=28/4; p<0.001) (FIG. 91, J). Thus,ischemic and excitotoxic insult had opposite effects on the expressionof Cx36- and Cx45-expressing GJs in the inner retina, consistent withtheir differential roles in mediating secondary cell death under thesetwo pathological conditions (FIG. 9E, F, K, L).

Example 6. Secondary Cell Death Via GJs Plays a Critical Role in theProgressive Loss of RGCs and ACs in Experimental Glaucoma

We next studied the role of secondary cell death in the progressive lossof RGCs and ACs in a mouse model of glaucoma and determined whetherpharmacologic or genetic blockade of GJs forms a novel approach forprotection of neurons in glaucomatous retinas. We posited thatGJ-mediated secondary cell death forms a critical mechanism in the lossof retinal neurons seen in glaucoma and thus blockade of GJs could offera novel strategy for protecting cells.

Experimental glaucoma was induced by intracameral injections ofpolystyrene microbeads in one eye of wild type (WT) mice, connexinknockout (KO) mice and their heterologous (Het) control littermates,with sham injection of the second eye as control. Injection ofpolystyrene beads significantly raised IOP from 11.4±0.3 to 21.9±0.5 mmHg, which remained elevated for at least 8 weeks following injection(FIG. 10A). We found no significant change in the RGC count from controllevels (50±1; p>0.1) within 1 week of bead injection. However, there wasa significant decrease in RGC count 4 weeks (40±3; p<0.05) and 8 weeksafter bead injection (33±1; p<0.001), a 20% and 36% population decrease,respectively (FIG. 10D). Overall retinal injury following bead injectionwas evidenced by an upregulation of GFAP in Muller cell processesspanning all retinal levels (FIG. 10B-C).

To test the role of secondary cell death via GJs in the loss of RGCs wenext blocked GJs before and after bead injection with MFA (50 μM).Blockade of GJs with MFA prevented the loss of RGCs by bead injection asevidenced by cell counts of 47±4 (p>0.1) at 8 weeks, comparable to thosein control retinas (FIGS. 11A-11F). Injection of MFA alone in controleyes had no detectable effect on RGC counts. FIGS. 11D-E show histogramscomparing the total cell (RGC+dAC) and RGC counts in the GCL ofmicrobead-injected retinas from WT mice untreated (D) or treated withMFA (E) to block GJs. Microbead injection clearly induced cell loss inthe GCL, but blockade of GJs prevented the loss. Thus, pharmacologicalblockade of GJs with MFA promotes RGC protection in experimentalglaucoma.

To determine whether the contribution of GJs to RGC death wasconnexin-specific, we induced experimental glaucoma in connexin KO mice(FIGS. 14A-14E). At 8 weeks after bead injections the RGC count was 40±1in Cx36 KOs, 43±1 for Cx45 KOs, and 49±3 for Cx36/45 dKO, indicating a41%, 59%, and 94% increase in survivability, respectively (FIG. 14A).GFAP expression was also significantly reduced in connexin KO mouseindicating an overall reduction in cell death (FIGS. 15A-15E). Since themajority of RGCs subtypes are coupled to (ACs) in the mouse retina, weexamined whether RGC could lead to AC death in experimental glaucoma. Weobserved a 20-30% loss of ACs in bead-injected WT mouse retinas, whichwas significantly prevented in Cx36 KO mice (FIGS. 12A-121). Ablation ofCx45 also prevented loss of coupled dACs (FIGS. 13A-13B). Microbeadinjection resulted in a loss of coupled dACs in wild-type, and a smallincrease in uncoupled dACs (FIG. 13A). However, there was no loss incoupled or uncoupled dACs in the Cx45−/− mouse retinas (FIG. 13B).

Our results provide clear evidence that GJ-mediated secondary cell deathplays a significant role in the propagation of cell loss in the adultretina seen under a number of different primary insult conditions.First, we observed that injection of the apoptotic agent CytC intoindividual RGCs and glia led to the exclusive loss of neighboringneurons to which they were coupled via GJs. Second, pharmacologicalblockade of GJs under excitotoxic and ischemic insult increased thesurvival of RGCs by approximately 70%, indicating that GJ-mediatedsecondary cell death plays a major role in cell loss. Further, underthese same insult conditions, blockade of GJs prevented nearly all ACdeath presumably by eliminating the propagation of toxic signals fromRGCs to which they were coupled. Finally, selective genetic ablation ofthe GJ subunits Cx36 or Cx45 found in the inner retina resulted in asignificant reduction in the loss of RGCs normally seen followingexcitotoxic or ischemic insult.

Studies of glaucomatous human retinas have reported an apparent delayedor secondary degeneration of amacrine cells subsequent to RGC cell loss.Consistent with this scenario is our present finding that CR- andCB-immunopositive amacrine cell lose due to excitoxicity or ischemiacould be largely blocked by GJ blockade. These data suggest that whileRGCs were most vulnerable under our experimental insult conditions, theloss of ACs was consequent to GJ-mediated bystander cell death. Thishypothesis is supported by our finding that ChAT-immunopositive ACs,which showed only minimal coupling to RGCs, were not significantlyaffected by excitotoxic or ischemic insult nor by disruption of GJs. Adownregulation of CR, CB, and ChAT have been reported in ischemicretinas; however, we found that ChAT-immunoreactive cells wereunaffected under our ischemic conditions and that MFA could maintaincell counts at control levels. These findings argue that the loss of CRand CB immunolabeling in ischemic retinas more likely reflected cellloss associated with GJ-mediated bystander cell death and not adownregulation of the protein markers.

In contrast to a role in secondary cell death, GJs have been reported tosometimes play a neuroprotective role. This raises the possibility thatthe role played by GJs in subserving cell death or neuroprotection maydepend on the nature of the primary insult.

Our results revealed another important difference between GJs in termsof their role in secondary cell death under different insult conditions.We found that whereas genetic ablation of Cx36, but not Cx45, couldsignificantly reduce cell loss under excitotoxic insult, ablation ofCx45, but not Cx36, protected cells from ischemic injury. These dataindicate that different cohorts of GJs, dependent on their connexinmakeup, subserve the bystander effect under different pathologicalconditions. Our results are the first to show that different cohorts ofGJs, based on the connexin they express, subserve secondary cell deathunder different primary insult conditions. Our immunolabeling datasuggests that this differential contribution of GJs under excitotoxicand ischemic conditions reflects changes in connexin protein expressionand manifestation. Under ischemic insult, we found that Cx36 protein wasaccumulated mainly in a perinuclear region of RGCs, but the normalpunctate immunolabeling in the IPL indicative of dendritic GJs wasabsent. A similar cytoplasmic internalization has been reported for Cx43in ischemic cardiac tissue, resulting from a dysfunction in Cx43trafficking linked to altered serine phosphorylation (Smyth et al.,Traffic; 15(6):684-99 (2014); Cone et al., J Biol Chem 289:8781-8798(2014)). Thus, in our experiments, while Cx36 protein was stillmanufactured under ischemic conditions, it appears to have not beeninserted in the membrane as functional GJs. In contrast, Cx45 punctatelabeling in the IPL appeared normal. These findings can explain whyischemic cell loss was reduced in the Cx45^(−/−) mouse retina, but notby ablation of Cx36, namely that Cx36-expressing GJs were alreadydisrupted by the insult. In contrast, we found that Cx45 immunolabelingunder excitotoxic insult was abnormal, with punctate labeling absentfrom the IPL, whereas Cx36 expression appeared normal. These datasuggest a downregulation of Cx45 under excitotoxicity and a lack offunctional GJs they constitute, which can explain why ablating Cx45 didnot reduce cell loss after NMDA-induced excitotoxicity.

The finding that bystander cell death in the retina is ultimatelyresponsible for the loss of most retinal neurons reveals that GJs form anovel, important target for neuroprotection. The identification of GJsas a therapeutic target is lent support by our ability to significantlylimit neuronal cell loss by blocking GJs with MFA administered afterischemic insult. Moreover, the finding that limited cohorts of GJs,specifically GJs expressing Cx36 and/or Cx45, are responsible forbystander death is propitious, as it shows that targeting of GJs, byinhibition of these specific connexins, has the potential for treatingretinal conditions associated with bystander cell death.

What is claimed is:
 1. A method of treating a condition of the retinacomprising administering an inhibitor of connexin 36 and/or an inhibitorof connexin 45 to a subject in need thereof.
 2. The method of claim 1,comprising administration of both an inhibitor of connexin 36 and aninhibitor of connexin
 45. 3. The method of claim 1 or 2 wherein saidcondition of the retina is selected from glaucoma, macular degeneration,retinitis pigmentosa, diabetic retinopathy and retinal ischemia.
 4. Themethod of any of claims 1-3 wherein said inhibitor or inhibitors isselected from an antisense polynucleotide directed to connexin 36 mRNA,an antisense polynucleotide directed to connexin 45 mRNA, andcombinations thereof.
 5. The method of claim 4 wherein said antisensepolynucleotide selectively binds the sequence of SEQ ID NO: 1 or SEQ IDNO:
 2. 6. The method of any of claims 3-5 wherein the antisensepolynucleotide is complementary to all of or a portion of connexin 36mRNA and/or connexin 45 mRNA.
 7. The method of claim 7 wherein saidantisense polynucleotide is the exact complement of all or a portion ofconnexin 36 mRNA and/or connexin 45 mRNA.
 8. The method of any of claims1-7 wherein said antisense polynucleotides hybridize to connexin 36 mRNAand/or connexin 45 mRNA with a melting temperature of greater than 20°C., 30° C. or 40° C. under physiological conditions.
 9. The method ofany of claims 1-3 wherein said inhibitor is a small molecule inhibitor.10. The method of claim 9 wherein said small molecule inhibitor isselected from 18-Beta-glycyrrhetinic acid (18Beta-GA) and meclofenamicacid (MFA).
 11. The method of any of claims 1-10, comprising repeatadministration of said inhibitor or inhibitors for a period of 1 week to1 year.
 12. The method of any of claims 1-11, wherein saidadministration is topical administration or intraocular injection.
 13. Apharmaceutical composition for treatment of a retinal conditioncomprising an inhibitor of connexin 36 and/or an inhibitor of connexin45.
 14. The composition of claim 13, wherein said composition comprisesan inhibitor of connexin 36 and an inhibitor of connexin
 45. 15. Thecomposition of claim 13 or 14, wherein said inhibitor or inhibitors areselected from an antisense molecule directed to connexin 36 mRNA, anantisense molecule directed to connexin 45 mRNA, and combinationsthereof.
 16. The composition of claim 13 or 14, wherein said compositioncomprises a small molecule inhibitor of connexin 36 and/or a smallmolecule inhibitor of connexin
 45. 17. The composition of any of claims13-16, formulated for topical administration to the eye.
 18. Use of thecomposition of any of claims 13-17 in the treatment of a condition ofthe retina.
 19. Use according to claim 18, wherein the condition of theretina is selected from glaucoma, macular degeneration, retinitispigmentosa, diabetic retinopathy and retinal ischemia.